Microtubules are polymers essential for cell morphogenesis, cell division and intracellular transport. Microtubules execute their diverse cellular roles by forming suprastructures with highly distinctive geometries: the radial cytoplasmic array, the short, highly parallel axonemal array, the spindle array or the tiled long axonal array. The microtubule cytoskeleton is a complex function of many unit operations, the individual actions of cytoskeletal regulators: nucleation, growth and shrinkage, severing and motor movement. Moreover, the microtubule itself is more than just a naive roadway for cellular components to transit along. Alpha and beta tubulins have multiple isoforms and are subject to highly diverse, abundant and evolutionarily conserved post-translational modifications that mark subpopulations of microtubules (Yu et al, 2015). Given the central role microtubules play in basic cellular processes, it is not surprising that microtubule regulators have been implicated in many human diseases, including cancers, cardiovascular disease, fungal, bacterial and viral infections, as well as neurodegenerative disorders such as Parkinson's, Alzheimer's and Amyotrophic lateral sclerosis. Our efforts concentrate on two families of microtubule regulators: microtubule severing enzymes and enzymes that post-translationally modify tubulin. Our research plan is highly interdisciplinary, integrating techniques and concepts from biophysics, structural, molecular and cell biology to answer two closely interdigitated questions: how is the structure of the microtubule locally perturbed when it is engaged by these regulators and how do these regulators affect microtubule architecture and dynamics at the cellular level? Perturbation of microtubule dynamics has emerged as a common theme in a variety of neurodegenerative diseases and our work has implications for the etiologies of all these disorders. In the last year we initiated several studies aimed at understanding the mechanistic underpinnings of the functions of microtubule post-translational modifications as well as continued our work on the mechanism of microtubule severing by spastin. Recent work from my group focused on the mechanism of action of the neuronal tubulin glutamylase TTLL7 (Garnham et al., 2015). Using a hybrid approach combining X-ray crystallography, cryo-electron microscopy, mass spectrometry and single molecule fluorescence we were able to visualize for the first time how a glutamylase of the TTLL superfamily of tubulin modification enzymes recognizes the microtubule and discriminates between soluble and polymeric tubulin as well as between &#945;- and &#946;-tubulin, ensuring preferential glutamylation of the &#946;-tubulin tail. The structure of the TTLL7 bound to the microtubule also allowed us to visualize for the first time the elusive C-terminal tails of &#945;- and &#946;-tubulin which are both engaged by TTLL7 as part of a tripartite microtubule recognition strategy that involves also a cationic microtubule binding domain that we found is universal in all tubulin glutamylases with autonomous activity. Removal of this domain in all glutamylases with autonomous activity reduces their activity to background level. My group has also been responsible in the last year for the development of novel methods for generating recombinant human tubulin as well as homogenous acetylated, glutamylated, or tyrosinated human tubulin and microtubules for in vitro assays (Vemu et al., 2014). The generation of differentially modified microtubules now enables a mechanistic dissection of the effects of tubulin post-translational modifications on the dynamics and mechanical properties of microtubules as well as the recruitment and behavior of motors and microtubule-associated proteins. Lastly, we are actively working on purifying to homogeneity and in biophysical quantities several other tubulin modification enzymes, both glutamylases and glycylases, to investigate their mechanism of action. We are currently also using these enzyme preparations to modify microtubules in vitro in order to investigate the effects of the introduced tubulin modification on microtubule dynamics and the recruitment and activity of motors and the microtubule severing enzymes spastin and katanin.